Iron deficiency (ID) remains one of the foremost world-wide nutrient deficiencies that affect brain development in the fetus and in children. ID affects at least 3 major aspects of early brain development: energy metabolism (especially in hippocampus (HC)), monoamine neurotransmitter homeostasis, and myelination, in turn potentially affecting behaviors such as recognition memory, procedural memory and speed of processing. In humans and dietary ID animal models, it is unclear whether structural and behavioral effects in the developing brain are due directly to a lack of iron interacting with important transcriptional, translational or post-translational processes or to indirect effects such as hypoxia (due to anemia) or perturbation of the homeostasis of other divalent cations important in brain development such as Zn, Cu or Mn. To circumvent these potential confounders and to directly assess iron's role in the development of the HC, we have generated two non-anemic genetic mouse models by conditionally altering the expression of two iron uptake transport proteins in area CA-1 of the HC. CA-1 is essential to recognition memory function and is altered metabolically, structurally and functionally in dietary ID. Model 1 relies on a CaMKinase II alpha-Cre-lox system to knock-out (KO) divalent metal transporter-1 (DMT-1), the major intracellular ferrous off-loading protein at embryonic day (E) 18 prior to CA-1 differentiation. This animal has been bred, is non-anemic and needs to be characterized from genotypic and iron phenotypic perspective. Model 2 uses a doxycycline induced (tetracycline transactivator) dominant-negative (DN) approach to functionally reduce the activity of transferrin receptor-1 (TfR-1), the major neuronal transmembrane iron uptake protein, in GA-1 pyramidal neurons. The parent lineages for this animal have been generated and are ready to be bred. The offspring will need to be genotypically and phenotypically characterized. The Specific Aims of this R-21 are to 1) confirm the absence of DMT-1 protein and mRNA, establish the hippocampal iron status and assess the behavioral phenotype of the conditional DMT-1 KO animal, and 2) to breed, confirm the mutation of TfR-1 in HC, establish the CA-1 iron status and assess the behavioral phenotype of the TfR-1 DN animal. The potential pay-offs of this high-risk, high gain proposal are models that define whether and, specifically, how iron is necessary for normal HC development at the molecular, protein and systems level. The models will be used to determine whether the lack of iron, per se, is responsible for the cognitive deficits in humans and animal models with dietary ID without the numerous confounding variables found in dietary models.